The goals of this proposal are to investigate the mechanism of genetic defects of the human pyruvate dehydrogenase complex (PDC), a key enzyme of energy metabolism, and to improve understanding of the relationship of normal structure and catalytic function of this enzyme and regulation of expression of the multiple genes responsible for its constituent proteins. Genetic defects of PDC are associated with congenital lactic acidosis, variably severe neurological disability, and, in many cases, early death. Through our previous efforts in this project we have identified 24 patients with PDC deficiency, the majority of whom have defects affecting the pyruvate dehydrogenase (E1) component. We developed specific immunological and cDNA probes for the various catalytic components of PDC and have used these to demonstrate heterogeneity of expression of E1 subunit proteins and mRNAs. We also generated complete primary nucleotide sequence's for the respective mRNAs and have used these to identify a point mutation affecting E1alpha. However, at the present time we are not able to account for the clinical heterogeneity of these disorders or their mode of inheritance and expression. Very little is known about regulation of the human E1alpha and E1beta genes or the specific roles of these subunits in catalysis. Our Specific Aims for continuation of this project are focused on the E1 component and include: i), characterization of genetic defects at the levels of protein, mRNA, and DNA; ii) organization and regulation of expression of the E1alpha and E1beta genes; and iii) characterization of the catalytic roles of each of these subunits. We propose to isolate and characterize the human E1beta structural gene and to characterize the promoter-regulatory regions of both the E1alpha and E1beta genes by expressing chimeric genes and analyzing DNA-protein interactions. We will individually express recombinant human E1alpha or E1beta proteins in order to investigate their respective catalytic roles and to analyze the interdependency of the three phosphorylation sites, using site-directed mutagenesis. In addition, we will locate specific amino acid residues in the alpha and beta proteins which are implicated in E1 catalysis by the combined use of chemical modification, isolation, and sequencing of modified peptides. Our multifaceted approach is designed to enhance understanding of the normal functions of these two E1 proteins and regulation of expression of their genes as well as the mechanisms accounting for the variable consequences resulting from mutations.